KR102272159B1 - Liver fibrosis mouse model without GOLGA2 gene - Google Patents

Abstract

The present invention relates to a liver fibrosis mouse model in which the GOLGA2 gene is knocked out, a composition for preparing the liver fibrosis mouse model, a method for preparing a liver fibrosis mouse model, and a screening method for inhibiting or treating liver fibrosis. When the GOLGA2 gene knockout liver fibrosis mouse model according to the present invention is used, there is an advantage that a model in which fibrosis is induced in the entire region of the liver due to a genetic mutation without serious lesion of the liver tissue can be used. In addition, the model can be used in various ways as a model for the study of the association between Golgi abnormal morphology, autophagy and liver fibrosis, and can be used as an animal model for screening a therapeutic agent for genetic-based liver fibrosis rather than chemical-based liver fibrosis.

Description

GOLGA2 gene knockout liver fibrosis mouse model {Liver fibrosis mouse model without GOLGA2 gene}

The present invention relates to a liver fibrosis mouse model in which the GOLGA2 gene is knocked out, a composition for preparing the liver fibrosis mouse model, a method for preparing a liver fibrosis mouse model, and a screening method for inhibiting or treating liver fibrosis.

Autophagy or self-eating is a process in which unnecessary or damaged intracellular proteins, lipids, and organelles are removed by lysosomes. Physiologically, the autophagy process does not simply mean elimination, but dynamically improves or improves cells. It plays an important role as a recycling system that supplies energy to maintain homeostasis and builds a foundation for new organelles. In relation to this autophagy, the Golgi apparatus is known to provide a double membrane (phagophore) that is generated in the early stage of autophagosome assembly.

On the other hand, liver fibrosis refers to a state in which damaged liver tissue is transformed into a fibrous tissue such as collagen, rather than being restored to normal hepatocytes, as part of a bioadaptation reaction accompanying chronic liver disease such as hepatitis. Although hepatic fibrosis is a bioadaptation reaction that occurs during the repair process of tissue damage, liver function is inevitably deteriorated because it replaces the liver tissue with fibrous tissue that cannot perform its own functions such as metabolism and bile secretion. The development of an appropriate therapeutic agent is an important task in the development of a new drug in that, when the liver fibrosis phenomenon is continuously repeated, it develops into liver cirrhosis and leads to death. However, until now, the mechanism of liver fibrosis itself has not been clearly elucidated, and a therapeutic agent with excellent efficacy has not yet been developed.

Existing models of liver fibrosis exist, but this is a chemical-based fibrosis model, which has a problem in that it causes serious lesions in the tissue area in contact with the chemical. In addition, the relationship between the Golgi apparatus, autophagy, and liver fibrosis and which genes are involved in these phenomena have not been elucidated, and specific studies are needed.

Accordingly, the present inventors completed the present invention by confirming that the GOLGA2 gene knockout mouse present in the cis-Golgi apparatus, which is the formation surface of the Golgi apparatus, for the first time during the study, causes the Golgi apparatus to collapse, induces autophagy, and induces liver fibrosis. did it

Accordingly, it is an object of the present invention to provide a liver fibrosis mouse model in which the GOLGA2 gene is knocked out, a composition for preparing the liver fibrosis mouse model, a method for preparing the liver fibrosis mouse model, and a screening method for a liver fibrosis inhibition or therapeutic agent.

In order to achieve the above object, the present invention provides a mouse model of liver fibrosis, in which the GOLGA2 gene is knocked out.

In addition, the present invention provides a composition for preparing a mouse model of liver fibrosis, comprising embryonic stem cells in which exons 1 to 26 of the GOLGA2 gene are deleted.

In addition, the present invention provides a method for preparing a mouse model of liver fibrosis, comprising the step of knocking out the GOLGA2 gene.

In addition, the present invention comprises the steps of (S1) treating a candidate substance in the liver fibrosis mouse model; and (S2) determining a liver fibrosis inhibition or therapeutic agent in the liver fibrosis mouse model treated with the candidate substance;

When the GOLGA2 gene knockout liver fibrosis mouse model according to the present invention is used, there is an advantage that a model in which fibrosis is induced in the entire region of the liver due to a genetic mutation without serious lesion of the liver tissue can be used. In addition, the model can be used in various ways as a model for studying the association between Golgi body abnormalities, autophagy and liver fibrosis, and can be used as an animal model for screening therapeutic agents for genetic-based liver fibrosis rather than chemical-based liver fibrosis. .

1 is a diagram showing the location of the GOLGA2 gene and a schematic diagram of the embryonic stem cells used to construct the GOLGA2 knockout mouse.
2 is a diagram showing the results of confirming that GOLGA2 protein is eliminated from Golgi by GOLGA2 knockout in liver cell lines.
3 is a diagram showing the results confirming that increased autophagy in GOLGA2 knockout liver cell line degrades fat droplets formed by free fatty acid (FFA).
4 shows GOLGA2 It is a diagram showing the result confirming that the expression of autophagy marker proteins SQSTM1, LC3I and LC3ll is increased in the knockout liver cell line.
5 is a diagram showing the results of genotyping of normal, heterozygous and GOLGA2 knockout mice.
6 shows GOLGA2 A diagram showing the results of selecting GOLGA2 knockout mice through the expression analysis of GOLGA2 mRNA in the tissues of the knockout mice.
7 shows normal, heterozygous and GOLGA2 It is a diagram showing a photograph comparing the size of knockout mice.
8 shows normal, heterozygous and GOLGA2 A diagram showing a size comparison graph of a knockout mouse.
9 shows normal, heterozygous and GOLGA2 It is a diagram showing a weight comparison graph of knockout mice.
10 shows male GOLGA2 It is a diagram showing the results of confirming the organ size reduction in knockout mice.
11 is a diagram showing the results of confirming that LC3B, an autophagy marker protein, is highly activated in specific cells of the liver in GOLGA2 knockout mouse liver tissue.
12 is GOLGA2 It is a diagram showing the result of confirming that the fat droplets were decomposed by autophagy in the liver through the electron microscope image analysis result of the knockout mouse liver.
13 is GOLGA2 A diagram showing the results of confirming that fibrotic lesions were found throughout the liver through histological analysis of the knockout mouse liver.
14 is GOLGA2 It is a diagram showing the result of confirming that smooth muscle actin (SMA) expression was increased through immunohistochemistry analysis of knockout mice liver.
15 is GOLGA2 It is a diagram showing the result of confirming that SMAD4, a sub-mechanical protein of the TGF-β signaling mechanism, was significantly increased through immunoblot analysis between knockout mice.
16 is a diagram showing a mechanism for preparing a mouse model of liver tissue fibrosis caused by knockout of the GOLGA2 gene.

The present invention provides a mouse model of liver fibrosis in which the GOLGA2 gene is knocked out.

The GOLGA2 gene of the present invention is a gene of NCBI Gene ID 99412 and is a gene belonging to chromosome 2 and exists in various species. Specifically, the GOLGA2 gene may be a polynucleotide comprising the nucleotide sequence of NCBI Reference Sequence NM_133852. In addition, the GOLGA2 gene is a gene present in the cis-Golgi apparatus, which is the formation surface of the Golgi apparatus.

The mouse model may be one in which the GOLGA2 protein is knocked out in the Golgi apparatus. As a result, the Golgi apparatus collapses and autophagy is induced. In addition, the mouse model may have increased autophagy in the liver. The mouse model may have increased expression of LC3B, an autophagy marker protein, in the liver.

In addition, the liver may be hepatic stellate cells, and fat droplets may be degraded due to an increase in autophagy in hepatic stellate cells. In addition, due to this, the hepatic stellate cells are transformed into fibrotic cells to cause fibrosis of the liver tissue.

The mouse model may be prepared from embryonic stem cells in which GOLGA2 gene exons 1 to 26 are deleted, but is not limited thereto. In addition, the embryonic stem cells may be injected into the blastocyst of a mouse.

The mouse model may be one in which SMAD4 protein is increased in the liver. The SMAD4 protein is a sub-mechanical protein of the TGF-β signaling mechanism, and since the increase in the TGF-β signaling mechanism is an important mechanism occurring in fibrosis, it can be confirmed that liver fibrosis is induced by confirming the increase of the SMAD4 protein.

Through the GOLGA2 gene knockout mouse model of the present invention, it can be confirmed that the GOLGA2 knockout causes the GOLGA2 protein to be knocked out in the Golgi apparatus, increases autophagy in the liver, and induces liver fibrosis. In addition, when the GOLGA2 gene knockout liver fibrosis mouse model according to the present invention is used, there is an advantage in that a model in which fibrosis is induced in the entire region of the liver due to a genetic mutation without serious lesion of the liver tissue can be used. In addition, the model can be used in various ways as a model for studying the association between Golgi body abnormalities, autophagy and liver fibrosis, and can be used as an animal model for screening therapeutic agents for genetic-based liver fibrosis rather than chemical-based liver fibrosis. .

In addition, the present invention provides a composition for preparing a mouse model of liver fibrosis, comprising embryonic stem cells in which exons 1 to 26 of the GOLGA2 gene are deleted. The composition may preferably be a medium composition, and the "culture media" refers to a medium capable of supporting cell growth and survival in vitro, and is suitable for culturing cells. Includes all media. The medium may contain essential and non-essential amino acids, vitamins, energy sources, lipids and trace elements necessary for minimal proliferation and/or survival of cells.

In addition, the present invention provides a method for preparing a mouse model of liver fibrosis, comprising the step of knocking out the GOLGA2 gene. The knockout may be performed by a conventional method known in the art, and preferably may be performed using a GOLGA2 CRISPR/Cas9 system, but is not limited thereto. If a mouse model of liver fibrosis is prepared through the step of knocking out the GOLGA2 gene, a model of liver fibrosis in which fibrosis is induced in the entire liver due to genetic mutation without serious lesion of liver tissue can be obtained. In addition, it is possible to obtain a genetic-based liver fibrosis model rather than a chemical-based liver fibrosis model.

In addition, the present invention comprises the steps of (S1) treating a candidate substance in the liver fibrosis mouse model; and (S2) determining a liver fibrosis inhibition or therapeutic agent in the liver fibrosis mouse model treated with the candidate substance;

The step (S1) is a step of treating a candidate substance in the liver fibrosis mouse model in which the GOLGA2 gene of the present invention is knocked out. The "candidate substance" is a substance expected to treat, prevent or ameliorate liver fibrosis, and is a chemical substance, oligonucleotide, peptide, gene, protein, food, formulation, compound, composition, drug, nutritional supplement, health care product or substances such as beverages, without limitation. In addition, the "treatment" may be a method of administering a candidate substance to the liver fibrosis mouse model of the present invention, but is not limited thereto. As the "administration method", an appropriate method among conventional administration methods such as oral administration, intravenous injection, subcutaneous administration, and intraperitoneal administration may be selected, and the dosage of the candidate substance may be determined according to the administration method, the weight and condition of the experimental animal, or in advance. It may be appropriately selected according to the experimental results, etc., but is not limited thereto.

The step (S2) is a step of determining a substance for inhibiting or treating liver fibrosis by selecting a substance confirmed to improve liver fibrosis among the candidate substances treated in the step (S1). The "screening" refers to a result of measuring the degree of liver fibrosis in the liver fibrosis mouse model of the present invention before the treatment of the candidate substance and the result of comparing the measurement result after the treatment of the candidate substance, the degree of liver fibrosis before the treatment of the candidate substance. This can be achieved by selecting a candidate material that has reduced .

If the screening method of the liver fibrosis inhibition or therapeutic agent using the liver fibrosis mouse model in which the GOLGA2 gene of the present invention is knocked out is used, the therapeutic agent can be screened using a model in which fibrosis is induced in the entire region of the liver due to genetic mutation. There is an advantage that In addition, it is possible to screen for therapeutic agents for genetic-based liver fibrosis rather than chemical-based liver fibrosis.

Hereinafter, the present invention will be described in more detail based on examples. The examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the invention is not limited by these examples according to the gist of the present invention.

The statistics of each experiment in the Examples were used ± S.D, and were reported as p < 0.05 (* or #), p < 0.01 (** or ##).

Experimental example 1. Liver Cell Line Culture and GOLGA2 knockout Cell line production

Chang cell line, a normal human hepatocyte, was added to Dulbecco's modified Eagle medium/high-glucose medium (WELGENE Inc.), fetal bovine serum 10%, antibiotics 10%, and Heps 20%, and cultured at _{37°C, 5% CO 2 in an incubator.}

In addition, the GOLGA2 knockout Chang cell line was constructed using the GOLGA2 CRISPR/Cas9 system.

Experimental example 2. GOLGA2 gene knockout mouse crafting

GOLGA2 1-26 exon-deleted embryonic stem cells were purchased from the Wellcome Trust Sanger Institute and injected into the blastocyst of C57B/L6J mice to obtain heterozygous progeny. GOLGA2 knockout mice were selected.

The PCR primers used for the genotyping were as follows. GOLGA2 KO-F (5'-CTTCGTATAATGTATGCTATACGAAGTTATGCTAGC-3') and Reg- GOLGA2 -R (5'-ACTGAGCCGGTGAAAACTTAGAAGC-3') were used to confirm GOLGA2 (-/-) knockout, and Mus GOLGA2 Exon 27 wt-F (5'-AAGACTGGCGGCCAAAGCC-3 ') and Reg- GOLGA2 -R was used to confirm the GOLGA2 (+ / +).

1 shows the location of the GOLGA2 gene and a schematic diagram of the embryonic stem cell used to construct the GOLGA2 knockout mouse. As shown in FIG. 1, the GOLGA2 gene belongs to chromosome 2 and GOLGA2 exon 1-26 of the used embryonic stem cell. It was confirmed that the number was deleted.

Experimental example 3. Immunity fluorescence ( Immunofluorescence ) analysis

After culturing the Chang cell line on an 8-well chamber slide for culture, it was fixed with 3% paraformaldehyde at 4°C for 10 minutes. After that, it was treated with 0.25% Triton® X-100 for 5 minutes and then blocked using 3% bovine serum albumin for 30 minutes, and the primary antibody was treated at 4°C for one day and then the fluorescently labeled secondary antibody was treated. did. After that, the liver tissue was sectioned with 4 μm of paraffin block and transferred to a slide, paraffin was removed and hydrated with xylene, and then boiled and cooled with retrieval buffer for 10 minutes to save antigen-antibody reaction. After blocking, the same method as above. was analyzed.

Experimental example 4. immunoblot (Western Blot) Analysis

^{For immunoblot analysis, 3X10 5} 3wells of each of the cultured Chang cell lines were placed on a 6-well plate, and the media was removed after 24 hours of incubation. After washing with 1X PBS, 20 mM Tris-HCl (pH 7.5), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% NP40, 10 mM beta-glycerophosphate, 50 mM fluorine Cells were lysed with a lysis buffer made of sodium fluoride, 0.2 mM sodium vanadate, 10 mM PNPP, 5 mM PMSF, and 10 μg/ml leupeptin. After measuring the protein concentration of the dissolved protein by Bradford protein assay (Bio-Rad, Hercules, CA), 40 ug was quantified, mixed with Laemmli sample buffer according to the concentration, boiled, loaded on SDS-PAGE, and transferred, and milk blocking (milk) blocking) and TBS-T washing, put the primary antibody, and reacted overnight. After TBS-T washing the next day, the Rabbit secondary antibody was incubated for 1 hour, followed by TBS-T washing 3 times for 20 minutes and developed as a film. Mouse liver tissue was frozen at very low temperature, crushed, and then obtained and analyzed in the same manner as above.

Experimental example 5. Histology and immunohistochemistry ( Immunohistochemistry ) characterization

The liver tissue of the GOLGA2 gene knockout mouse was fixed in 10% formalin and then paraffin-embedded, and the tissue block was transferred to a silane-coated slide after sectioning at 4 μm. For histological analysis, the deparaffinized tissue slides were stained with hematoxylin and eosin (H&E) or with Sirius Red reagent to observe collagen deposition. In addition, for immunohistochemistry analysis, the deparaffinized tissue sections were boiled in retrival buffer and then treated with 5% hydrogen peroxide for 15 minutes to remove endogenous peroxidase, followed by washing with TBS buffer containing tween 20. and blocked for 1 hour. Subsequently, after treatment with the primary and secondary antibodies, it was washed again, and after color development with DAB substrate solution, it was quickly washed in sterilized water. For counter staining, it was stained with hematoxylin. Thereafter, it was observed under a tissue microscope through alcoholization and mounting.

Experimental example 6. Transmission electron microscope, TEM ) analysis

For TEM analysis, liver tissue was fixed overnight at 4°C with 2.5% glutaraldehyde buffer containing 1% osmium tetroxide, followed by alcoholization, and propylene oxide epon resin was applied to the tissue. After infiltration and hardening at 70°C overnight, ultra-thin sections of 40 nm were placed on a copper grid and observed using a JEM1010 TEM (JEOL).

Example 1. In liver cell lines GOLGA2 knock out autophagy confirm that it causes action

In order to confirm the effect of GOLGA2 knockout on the GOLGA2 knockout liver cell line prepared in Experimental Example 1, immunofluorescence analysis was performed, and the results are shown in FIG. 2 .

2, the time sikyeoteul knockout GOLGA2 the gene in the liver cell line GOLGA2 protein was confirmed by checking the eopeojim in the Golgi, GOLGA2 four GOLGA2 Golgi protein knockout in cell lines out. Blue represents the nucleus.

In addition, in order to confirm that autophagy was induced in GOLGA2 knockout liver cell line, normal hepatocyte cell line (Chang) and GOLGA2 knockout hepatocyte cell line were treated with 250 μM free fatty acid (FFA) for 24 hours, followed by triglyceride. Fat droplets were identified by (neutral lipid) staining, and the results are shown in FIG. 3 . In FIG. 3, blue indicates a nucleus.

As shown in FIG. 3 , it was confirmed that fat droplets formed by FFA were degraded due to an increase in autophagy in the GOLGA2 knockout liver cell line. Therefore, it was confirmed that the size of the fat droplets was reduced due to the increase in autophagy in the GOLGA2 knockout hepatocyte line.

In addition, to confirm the expression of autophagy marker proteins SQSTM1, LC3I and LC3ll in the GOLGA2 knockout liver cell line, an immunoblot analysis was performed, and the results are shown in FIG. 4 .

As shown in FIG. 4 , it was confirmed that SQSTM1, LC3I and LC3II, known as autophagy marker proteins, increased in the GOLGA2 knockout liver cell line.

Therefore, it was confirmed from the above results that when the GOLGA2 gene was knocked out, the GOLGA2 protein was knocked out in the Golgi apparatus and autophagy increased.

Example 2. GOLGA2 gene knockout Mouse Characterization

In order to examine the characteristics of the GOLGA2 gene knockout mouse prepared in Experimental Example 2, it was analyzed as follows.

* Example 2-1. Genotyping

Polymerase chain reaction (PCR) was performed to select GOLGA2 gene knockout mice prepared in Experimental Example 2, and the genotypes of normal, heterozygous and GOLGA2 knockout mice were analyzed through PCR. indicated.

As shown in FIG. 5 , as a result of confirming GOLGA2 knockout mice through PCR, mice in which only the GOLGA2 KO band was observed were selected as GOLGA2 knockout mice.

Example 2-2. mRNA Expression analysis

In order to confirm that all of the GOLGA2 gene was knocked out in the tissues of the selected GOLGA2 knockout mice, mRNA expression was analyzed, and the results are shown in FIG. 6 .

As shown in FIG. 6 , it was confirmed that GOLGA2 was completely knocked out in all organs.

Example 2-3. Mouse size comparison analysis

Pictures and graphs comparing the sizes of normal, heterozygous and GOLGA2 knockout mice are shown in FIGS. 7 and 8, respectively.

As shown in FIG. 7 , it was confirmed that the size of the GOLGA2 knockout mouse was smaller than that of the normal mouse, and in particular, the size was smaller in males.

In addition, as shown in the graph of FIG. 8 , it was confirmed that only GOLGA2 knockout mice significantly decreased in size compared to normal, regardless of sex (n=3).

Example 2-4. Mouse weight comparison analysis

The weights of normal, heterozygous and GOLGA2 knockout mice were measured, and a comparison graph is shown in FIG. 9 .

As shown in FIG. 9 , it was confirmed that there was no change in the body weight of females in each group, and the weights of heterozygous and GOLGA2 knockout mice in males significantly decreased compared to normal mice (n=3).

Example 2-5. knockout Analysis of organ size in mice

The organ sizes of GOLGA2 knockout mice were compared and shown in FIG. 10 .

As shown in FIG. 10 , it was confirmed that the organ size of male GOLGA2 knockout mice was reduced compared to that of normal male mice.

Example 3. GOLGA2 knockout in the mouse liver GOLGA2 knock out autophagy Confirm that it causes action

Immunofluorescence staining analysis was performed to confirm whether GOLGA2 knockout induced autophagy in the liver through the GOLGA2 gene knockout mouse prepared in Experimental Example 2, and the results are shown in FIG. 11 .

As shown in FIG. 11 , it was confirmed that LC3B staining, an autophagy marker protein, was highly activated in specific cells of the liver in GOLGA2 knockout mouse liver tissue. In addition, the number was analyzed to be less than 10% of the whole liver, and it was confirmed that it was highly likely to be hepatic macrophage or hepatic stellate cells.

In addition, the results of electron microscope image analysis of GOLGA2 knockout mice are shown in FIG. 12 . In Fig. 12, Nu denotes a nucleus, M denotes mitochondria, G denotes the Golgi apparatus, and LD denotes a fat droplet.

As shown in Fig. 12, normal hepatocytes have normal morphology of fat droplets and Golgi bodies, but GOLGA2 Fat droplets and Golgi bodies were not observed in hepatocytes of knockout mice, and it was confirmed that mitochondria were surrounded by a double membrane. This is a phenotype found in mitochondrial autophagy, through which GOLGA2 It was confirmed that fat droplets were degraded by autophagy in the liver of knockout mice.

Therefore, it was confirmed through the above results that GOLGA2 knockout activates autophagy in the liver of GOLGA2 knockout mice.

Example 4. GOLGA2 knockout through the mouse In liver fibrosis GOLGA2 knock out Check the impact

Through the GOLGA2 gene knockout mouse prepared in Experimental Example 2, a histological analysis was performed to confirm the effect of GOLGA2 knockout on liver fibrosis in the liver, and the results are shown in FIG. 13 .

As shown in FIG. 13 , it was confirmed that the density of hepatocytes around the hepatic duct of GOLGA2 knockout mice was increased during H&E staining. In addition, it was confirmed that fibrotic lesions were found throughout the liver in Sirius red staining (black arrow in FIG. 13 ).

This result is the result of confirming that fibrosis occurs in the entire liver due to genetic factors, unlike the confirmation of liver fibrosis around the liver duct by the conventional compound, carbon tetrachloride.

In addition, the results of immunohistochemistry analysis of GOLGA2 gene knockout mice are shown in FIG. 14 .

As shown in Fig. 14, GOLGA2 It was confirmed that the expression of smooth muscle actin (SMA), an indicator protein of fibrosis, was increased around the liver duct of knockout mice.

In addition, the results of immunoblot analysis between GOLGA2 gene knockout mice are shown in FIG. 15 .

As shown in Figure 15, GOLGA2 It was confirmed that SMAD4, a sub-mechanical protein of the TGF-β signaling mechanism, significantly increased in the liver of knockout mice. Since this increase in the TGF-β signaling mechanism is an important mechanism for fibrosis, it was confirmed that GOLGA2 knockout also causes liver fibrosis.

Example 5. GOLGA2 gene knocked out liver tissue Establishment of a fibrotic mouse model

Through the above examples, it was confirmed that deletion of the GOLGA2 gene, a Golgi protein, induces autophagy , and intracellular lipids are degraded by autophagy, especially in hepatic stellate cells containing a lot of lipids. In addition, it was confirmed that due to this, the hepatic stellate cells were transformed into fibrotic cells to cause fibrosis of the liver tissue. Through this, a mouse model of liver tissue fibrosis in which the GOLGA2 gene was knocked out was finally established, which is shown in FIG. 16 .

Claims (5)

A composition for preparing a mouse model of liver fibrosis comprising embryonic stem cells in which GOLGA2 gene exons 1 to 26 are deleted.
According to claim 1,
The composition for preparing the mouse model,
A composition for preparing a mouse model of liver fibrosis, characterized in that it knocks out the GOLGA2 protein from the Golgi apparatus of hepatocytes of the mouse model.
According to claim 1,
The composition for preparing the mouse model,
A composition for preparing a mouse model of liver fibrosis, characterized in that it enhances autophagy in the liver of the mouse model.
4. The method of claim 3,
The liver,
A composition for preparing a mouse model of liver fibrosis, characterized in that it is hepatic stellate cells.
According to claim 1,
The composition for preparing the mouse model,
A composition for preparing a mouse model of liver fibrosis, characterized in that it enhances the expression of LC3B protein or increases the SMAD4 protein in the liver of the mouse model.

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